The retinas of most adult vertebrates cannot regenerate neurons. In contrast, adult zebrafish regenerate all classes of retinal neurons including the photoreceptors. For example, light-induced photoreceptor cell damage of the adult zebrafish retina induces the Muller glia to reenter the cell cycle and generate neuronal progenitor cells. These Muller glial-derived progenitors migrate to the outer nuclear layer and differentiate into new rods and cones. In contrast to zebrafish, human retinal Muller cells cannot regenerate any retinal cell class. By understanding the genetic and biochemical basis of Muller glia-based neuronal regeneration in the light-damaged zebrafish retina, we may identify approaches to stimulate a similar regeneration response in the human retina. This could yield a strategy to restore vision to individuals with genetic and environmental forms of blindness. We determined Pax6 is expressed in the new neuronal progenitor cells shortly after being generated from the Muller glial cells. To determine the role of the Pax6 protein in these progenitors during regeneration, we need to disrupt Pax6 expression. Unfortunately, Pax6 is required for retinal development and disruption of Pax6 expression during early zebrafish development will prevent eye formation. Thus, we developed an electroporation technique to deliver antisense morpholinos into the progenitor cells of the regenerating adult retina and conditionally block Pax6 protein expression. Our preliminary results demonstrate we can block Pax6 expression in the neuronal progenitor cells, which prevents their proliferation. We will demonstrate the utility of this new technique and determine the role of Pax6 in progenitor cell proliferation and photoreceptor regeneration. We will also begin to identify Pax6 transcriptional targets that may play roles during the retinal regeneration response. [unreadable] [unreadable] Relevance: While several candidate genes were identified that may play important roles for retinal regeneration in zebrafish, a method must be developed to test the function of these candidate genes/proteins. Standard forward genetic approaches will not be amenable for this analysis because most of these genes are also required for retinal development. We describe a morpholino electroporation technique to conditionally knockdown the expression of the desired protein in the adult retina. To demonstrate the power of this method, we will examine the speculative function of the Pax6 protein for zebrafish retinal regeneration.Many vision diseases cause loss of human photoreceptors that cannot be regenerated. Robust photoreceptor regeneration in zebrafish involves expressing the Pax6 protein as the Muller glial cells divide and produce neuronal progenitor cells, which then renew the lost photoreceptors. Understanding the role of Pax6 in zebrafish retinal regeneration may provide clues as to why the human Muller glial cells cannot regenerate photoreceptors, and ultimately lead to the ability to induce endogenous Muller glial cells to regenerate photoreceptors in the human retina. [unreadable] [unreadable] [unreadable]